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An ELISA method to assess HDAC inhibitor-induced alterations to P. falciparum histone lysine acetylation
International Journal for Parasitology: Drugs and Drug Resistance ( IF 4 ) Pub Date : 2020-11-02 , DOI: 10.1016/j.ijpddr.2020.10.010
Eva Hesping 1 , Tina S Skinner-Adams 1 , Gillian M Fisher 1 , Thomas Kurz 2 , Katherine T Andrews 1
Affiliation  

The prevention and treatment of malaria requires a multi-pronged approach, including the development of drugs that have novel modes of action. Histone deacetylases (HDACs), enzymes involved in post-translational protein modification, are potential new drug targets for malaria. However, the lack of recombinant P. falciparum HDACs and suitable activity assays, has made the investigation of compounds designed to target these enzymes challenging. Current approaches are indirect and include assessing total deacetylase activity and protein hyperacetylation via Western blot. These approaches either do not allow differential compound effects to be determined or suffer from low throughput. Here we investigated dot blot and ELISA methods as new, higher throughput assays to detect histone lysine acetylation changes in P. falciparum parasites. As the ELISA method was found to be superior to the dot blot assay using the control HDAC inhibitor vorinostat, it was used to evaluate the histone H3 and H4 lysine acetylation changes mediated by a panel of six HDAC inhibitors that were shown to inhibit P. falciparum deacetylase activity. Vorinostat, panobinostat, trichostatin A, romidepsin and entinostat all caused an ~3-fold increase in histone H4 acetylation using a tetra-acetyl lysine antibody. Tubastatin A, the only human HDAC6-specific inhibitor tested, also caused H4 hyperacetylation, but to a lesser extent than the other compounds. Further investigation revealed that all compounds, except tubastatin A, caused hyperacetylation of the individual N-terminal H4 lysines 5, 8, 12 and 16. These data indicate that tubastatin A impacts P. falciparum H4 acetylation differently to the other HDAC inhibitors tested. In contrast, all compounds caused hyperacetylation of histone H3. In summary, the ELISA developed in this study provides a higher throughput approach to assessing differential effects of antiplasmodial compounds on histone acetylation levels and is therefore a useful new tool in the investigation of HDAC inhibitors for malaria.



中文翻译:

一种评估 HDAC 抑制剂诱导的恶性疟原虫组蛋白赖氨酸乙酰化改变的 ELISA 方法

疟疾的预防和治疗需要多管齐下的方法,包括开发具有新型作用方式的药物。组蛋白去乙酰化酶 (HDAC) 是一种参与翻译后蛋白质修饰的酶,是治疗疟疾的潜在新药靶点。然而,由于缺乏重组恶性疟原虫HDACs 和合适的活性测定,使得针对这些酶的化合物的研究具有挑战性。目前的方法是间接的,包括通过蛋白质印迹评估总脱乙酰酶活性和蛋白质超乙酰化。这些方法要么不允许确定不同的化合物效应,要么受到低通量的影响。在这里,我们研究了斑点印迹和 ELISA 方法作为新的、更高通量的检测方法来检测组蛋白赖氨酸乙酰化变化恶性疟原虫寄生虫。由于发现 ELISA 方法优于使用对照 HDAC 抑制剂伏立诺他的斑点印迹分析,因此它被用于评估由一组六种 HDAC 抑制剂介导的组蛋白 H3 和 H4 赖氨酸乙酰化变化,这些抑制剂显示出抑制恶性疟原虫脱乙酰酶活性。使用四乙酰赖氨酸抗体,伏立诺他、帕比司他、曲古抑菌素 A、罗米地辛和恩替司他都导致组蛋白 H4 乙酰化增加约 3 倍。Tubastatin A 是唯一测试的人类 HDAC6 特异性抑制剂,也引起 H4 过度乙酰化,但程度低于其他化合物。进一步调查显示,除 tubastatin A 外的所有化合物均导致单个 N 端 H4 赖氨酸 5、8、12 和 16 的过度乙酰化。这些数据表明 tubastatin A 影响恶性疟原虫H4 乙酰化与测试的其他 HDAC 抑制剂不同。相比之下,所有化合物都会引起组蛋白 H3 的过度乙酰化。总之,本研究中开发的 ELISA 提供了一种更高通量的方法来评估抗疟原虫化合物对组蛋白乙酰化水平的不同影响,因此是研究疟疾 HDAC 抑制剂的有用新工具。

更新日期:2020-12-03
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